New insights into the diversity of cryptobenthic Cirripectes blennies in the Mascarene Archipelago sampled using Autonomous Reef Monitoring Structures (ARMS)

Abstract Autonomous Reef Monitoring Structures (ARMS) are artificial mini‐reefs designed for standardized sampling of sessile and small motile cryptobenthic organisms. ARMS are also effective for collecting small cryptobenthic fishes, such as the combtooth blennies of the genus Cirripectes. Recent studies discovered several Cirripectes species endemic to islands or archipelagos, in spite of the generally broad distributions of tropical and subtropical blennies. Thus, to evaluate the diversity and distribution of Cirripectes species in the Mascarene Archipelago, a little‐studied region but an important biodiversity hotspot, complete mitochondrial genomes, and nuclear rhodopsin genes were sequenced for 39 specimens collected with ARMS deployed on outer reef slopes at Reunion and Rodrigues islands. Mitochondrial COI sequences were analyzed to integrate these specimens within the largest dataset of publicly available sequences. Three species were found in the Mascarene Archipelago, Cirripectes castaneus, Cirripectes randalli, and Cirripectes stigmaticus. C. castaneus and C. stigmaticus both have an Indo‐Pacific distribution with several haplotypes shared among distant localities. In agreement with the literature, C. randalli shows a small‐range endemism restricted to the Mascarenes. We confirmed the presence of C. castaneus, C. randalli, and C. stigmaticus in Rodrigues, and the presence of C. stigmaticus in Reunion. This study contributes to filling the gaps in taxonomic and molecular knowledge of the reef cryptobiome in the South‐West Indian Ocean, and provides the first complete mitogenomes for the genus, a crucial step for future molecular‐based inventories (e.g., eDNA).


| INTRODUC TI ON
Understanding coral reef ecosystem functioning requires knowledge of the distribution and abundance of reef-associated fishes.
Due to their accessibility to visual observation, the larger reef fishes have been intensively studied for decades (Knowlton et al., 2010).
Although their diversity and taxonomy appeared relatively well resolved (Allen, 2015;Fisher et al., 2015;Mora et al., 2008), molecular studies revealed extensive hidden diversity (Hubert et al., 2017;Steinke et al., 2009). The smaller taxa present additional challenges, as they are inherently more difficult to find and identify, and are therefore often omitted from visual surveys and collections Brandl et al., 2018;Pearman et al., 2018).
Although they are often overlooked, their distinctive demographic dynamics may make them a cornerstone of ecosystem functioning in modern coral reefs .
Cryptobenthic reef fishes are small, bottom-dwelling, morphologically, or behaviorally cryptic species. They comprise families such as combtooth blennies (Blenniidae), gobies (Gobiidae), triplefins (Tripterygiidae), and cardinalfishes (Apogonidae). Despite being the ocean's smallest vertebrates, they contribute disproportionately to coral reef food webs through their high abundance, rapid somatic growth, and high predation mortality, producing almost 60% of reef fish biomass consumed within the ecosystem .
Furthermore, cryptic fishes represent approximately 10% of vertebrate diversity on coral reefs and can exhibit high levels of endemism Brandl et al., 2018).
The genus Cirripectes Swainson, 1839 (Family Blenniidae, Order Blenniiformes) comprises 24 recognized species of combtooth blennies, broadly distributed in the Indo-Pacific from East Africa to Rapa Nui in the eastern Pacific (Hastings & Springer, 2009;Hoban & Williams, 2020;Williams, 1988). Currently, 14 species are recorded with COI DNA sequences in the BOLD database (Ratnasingham & Hebert, 2007), plus three genetically divergent groups that possibly represent not yet described new species. Most Cirripectes species are smaller than 100 mm. They are herbivorous and/or detrivorous cryptobenthic teleosts that primarily inhabit rocky or coral substrates in shallow (<5 m depth) high-surge fore reef habitats (Williams, 1988). However, individuals of Cirripectes matatakaro and Cirripectes castaneus may be encountered deeper (more than 20 m and over 32 m depth, respectively; Williams, 1988, Hoban & Williams, 2020. Species show considerable variation in geographic range sizes, from small area endemism (e.g., Cirripectes heemstraorum endemic to the East coast of South Africa at Cape Vidal; Williams, 2010) to Indo-Pacific-wide distributions (e.g., C. castaneus; Williams, 1988). Blennies' eggs are demersal and attached to the substratum with a filamentous, adhesive pad or pedestal (Breder & Rosen, 1966;Watson, 2009). Larvae are planktonic and abundant in shallow, coastal waters (Watson, 2009). While Cirripectes specimens are common in museum collections, incomplete knowledge of sexual dimorphism and geographic color variations, combined with a lack of adequate species identification keys, have resulted in numerous misidentifications and undetected cryptic species (Williams, 2010).
In many cases, color morphs were considered distinct species, even though sexual polychromatism has been described for several Cirripectes species (Williams, 1988).
Cirripectes filamentosus, Cirripectes polyzona, Cirripectes quagga, and C. stigmaticus occur throughout the Indo-Pacific, while the distribution of C. auritus and C. castaneus ranges from East Africa to the western Pacific. C. gilberti has a more restricted distribution and occurs throughout the Indian Ocean (Williams, 1988). However, recent studies discovered several Cirripectes species endemic to islands or archipelagos (Delrieu-Trottin et al., 2018;Hoban & Williams, 2020). For the Mascarenes, only C. randalli is known to have a distribution limited to the archipelago (Williams, 1988).
Ecological and geographical distributions of these species are summarized in Table 1.
Cryptobenthic fishes are often under-sampled due to their hidden habits. Therefore approaches focused on sampling small and cryptobenthic fauna, such as artificial mini-reefs ARMS, represent alternative sampling techniques. ARMS, for Autonomous Reef Monitoring Structures, are stacks of nine PVC plates spaced at a 12 mm distance, designed to mimic the complexity of coral reef habitats. Each ARMS represents slightly over 4.5 L of habitat volume. Affixed to the seabed, they are left to be colonized by a diversity of marine species, then collected and dismantled to study the associated biota (see Zimmerman & Martin, 2004 for more details).
To evaluate the diversity and distribution of Cirripectes species in the Mascarene Archipelago, a little-studied region with high endemism and source of type material for this genus, we reconstructed the phylogeographic relationships within Cirripectes collected using ARMS. We conducted a multi-marker approach by sequencing mitochondrial and nuclear genes and integrated newly collected specimens within the largest dataset of publicly available sequences. We sequenced complete mitochondrial genomes as part of the current effort to complete the inventory of teleosts in French territories led by the Muséum national d'Histoire naturelle (MNHN). Having available complete mitogenomes enables the construction of more robust phylogenies, but the current paucity of mitogenomes in public databases makes such analyses premature. Finally, we explored the nucleotide divergences between Cirripectes species found in the Mascarene Islands to assess the performance of current mini-barcodes used to detect teleosts species in eDNA studies. the entire mitochondrial genome and the partial retro-rhodopsin nuclear gene (Rh193 and Rh1039r; Chen et al., 2003). The mitogenome was amplified in three overlapping fragments. The first fragment from the end of the 16S to the end of COI was amplified with 16SAR (Kocher et al., 1989) and MtH7061 (Hinsinger et al., 2015). The second fragment from the beginning of COI to the end of ND4 was amplified with F5231cha (Hinsinger et al., 2015) and MtH11944 (Hinsinger et al., 2015). Additionally, we developed three new primers, R11944cha 5′-CATAG CTN CTA CTT GGA TTT GCACCA-3′ and two specific primers designed for the genus Cirripectes: F5231Cirri 5′-TAGRC AGG CAG GCC TCG ATC CTRCA-3′ and R11944Cirri 5′-CATAG TTT CTG CTT GGA GTT GCACCA-3′ to improve amplification success. The third fragment from ND5 to the end of 16S was amplified with MtL11910 (Hinsinger et al., 2015) and 16SBR (Kocher et al., 1989). The amplicons were pooled with other PCR amplicons following Hinsinger et al. (2015) for cost efficiency. Library preparation followed Meyer and Kircher (2010)

| Sequences processing and public sequences retrieval
Reads were processed with Geneious Prime 2019.2.3. Paired-end reads were merged, the primers were used as barcodes to recover the fragment ends and the merged reads were de novo assembled. The resulting contigs were checked in BOLD and Genbank databases before being used as references for elongation through TA B L E 1 Cirripectes species reported from the Mascarene Archipelago with their ecological and geographical distributions.

Species Habitat Depth Polymorphisms Distribution
C. auritus (Carlson, 1981) (Williams, 1988) Rocky and coralline substrates <8 m S Indian Ocean C. polyzona (Bleeker, 1868) Algal ridges and crests between surge channels of exposed seaward reefs Usually <3 m; <20 m S Indo-Pacific C. quagga (Fowler & Ball, 1924) Algal ridges and crests between surge channels of exposed seaward reefs <10 m; max 19 m S + G* Indo-Pacific C. randalli (Williams, 1988) Coral patches in surge channels of rocky reefs with light surf <8 m S Mascarenes C. stigmaticus (Strasburg & Schultz, 1953) Upper edge of seaward reef slopes. Adults inhabit coastal reef flats with rich corals and algae. Among Acropora and Pocillopora corals of wave-swept algal ridges <20 m S + G Indo-Pacific Note: The types of polymorphism were indicated as 'S' for sexual and 'G' for geographical. Species with more than one sympatric sexual color pattern are indicated by *. Information was synthetized from Williams (1988), Letourneur et al. (2004), and Allen et al. (2013).
repeated mapping of reads with a maximum of 1% mismatch and three bases gap allowed. Mapping was repeated until no further reads could be mapped. Complete linear mitochondrial consensuses were transformed into circular sequences and overlapping sections were manually inspected and adjusted. Last, reads were mapped back against the obtained circular sequences to check coverage and final assembly. The mitogenome sequences were annotated using online MitoFish (Iwasaki et al., 2013). Sanger sequences were assembled and checked in Geneious Prime

(2019).
To increase taxonomic and spatiotemporal coverage, our datasets were extended with publicly available sequences from BOLD and/or GenBank ( Figure 1; ESM 1). In some instances, sequences were renamed according to the conclusions of the papers in which they were published, even if the names were not corrected in the database: sequences within BOLD BIN:AAU0601 were renamed from C. castaneus to C. randalli (Hoban & Williams, 2020), and MH932003 to MH932007 were renamed from C. alboapicalis to C. patuki sensu Delrieu-Trottin et al., 2018(Delrieu-Trottin et al., 2018. Two sequences, GBMNB4802-20 and KX223895.1, respectively from BOLD and Genbank, probably belong to a different genus and were removed from the analyses.

| Molecular and phylogenetic analyses
All analyses were performed on three datasets: (i) mitochondrial cytochrome oxidase I (further called COI dataset; N = 296; ESM 1), Sequences of each marker were aligned separately via Muscle 3.8.425 (Edgar, 2004), implemented in Geneious Prime 2019.2.3 using default parameters, and manually trimmed to maximize the shared length among the sequences (COI = 506 bp, Rho = 737 bp).
For the mt dataset, complete mitogenomes were aligned and translated into protein to check the codon position for the coding genes. The two tRNA and 13 CDS were kept according to coding position and overlapping CDS portions were trimmed at the end to prevent a shift in the translation frame.

| Sliding window analyses
Sliding window analyses were performed to explore nucleotide divergence between Cirripectes species found in the Mascarene Islands and to determine the performance of current minibarcodes used to detect teleosts species in eDNA (MiFish [163-185 bp;Miya et al., 2015]; Teleo [65 bp; Valentini et al., 2016]).
These analyses were performed using the R package "SPIDER" 1.1.2 (Brown et al., 2012) with a window size of 160 and 65 bp for the MiFish and the Teleo markers, respectively, and with a step size of 1 bp. Divergence analyses were also performed on complete COI and partial COI fragments from Geller et al., 2013 which are mostly used for metabarcoding of broad taxonomic groups.

| Sequence analysis
The were informative) than the first (11.31%) and the second (0%) codon positions.

| Phylogenic reconstruction of Cirripectes
For the COI dataset, the nucleotide substitution model with codon partition had less good fit than the single model; hence, the GTR + F + I + G4 model was selected for the analysis (Appendix S4).
For the mitochondrial concatenated dataset sequences, the bestfit partitioning scheme was GTR + I + G4. For rhodopsin, different partition models were used. While TVM + F + G was the best-fit partitioning scheme for the first codon position, this scheme was unfortunately not implemented in BEAST. However, it was equivalent to the GTR + F + I + G4 model with fixed AG rate parameters (1.0; Bagley, 2018). For the second and third codon positions, F81 + F + G4 and GTR + F + G4 were respectively selected.
Maximum-likelihood and BI-based phylogenetic reconstructions for COI resulted in tree topologies with marked similarities ( Figure 2). For the 17 species in the dataset, the COI trees recovered well-supported clades within the genus. However, the branching order of these clades presented differences among methods.
Clades 6 and 7 were sister species in the BI analysis, while clade 6 was closer to clade 8 within the ML approach. These incongruent

| Molecular species delimitation
Based on the COI dataset, the ASAP molecular species delimitation approach divided the sequences into 16 groups, while the GMYC and BIN subdivided the COI dataset into 17 groups, and mPTP subdivided into 19 groups ( Figure 2; ESM 1). For the sequences from specimens identified as C. filamentosus and Cirripectes chelomatus, GMYC, BIN, and mPTP methods divided sequences according to geographic origin (the western Indian and western Pacific oceans, respectively) and distinct morphological characters (Williams, 1988).
The mPTP method was the only one that subdivided specimens identified as Cirripectes matakaro into two groups, as was the case for those identified as Cirripectus variolosus. These two subdivisions were not supported by the other methods and they will not be considered further here. Therefore, a final delimitation scheme was established based on a majority-rule consensus among the dif-

| Sequence variability and haplotype relationship
Nucleotide diversity for the COI marker was low and ranged from 0.000 to 0.014 ( to the insufficient number of sequences (N = 1; Table 2). For rhodopsin, the AMOVA results also supported the above species grouping (for species present in the dataset: Fst = 0.86; p < .001; Appendix S7).
The haplotype networks based on COI sequences were computed for each of the 17 species from the molecular delineation analyses ( Figure 2  dataset generated similar but less detailed information in terms of haplotype diversity, due to the lack of representation by available sequences (Appendix S10). No haplotypes were shared by species among the specimens studied.

| Extension of DNA barcode library and mitochondrial genome
The 24 complete mitochondrial genome sequences produced in this study belong to three species, C. castaneus (N = 19), C. stigmaticus (N = 4), and C. randalli (N = 1). These represent the first complete mitogenome sequences for the genus Cirripectes. Appendix S11.1), respectively. More detailed information is provided in Appendix S11.

| Sliding window analyses
Two pairwise comparisons between the most closely related species pair (C. castaneus with C. stigmaticus) and the more distant one (C. stigmaticus with C. randalli), were chosen for sliding window analysis. The distribution of divergent sites is shown in Figure 3.

| Cirripectes species of Reunion and Rodrigues
Following the results of the delimitation analyses, from the 34 new COI sequences generated in this study, 28 were assigned to C. castaneus, 4 to C. stigmaticus, and two to C. randalli. Among the 26 specimens sequenced for rhodopsin, 22 were assigned to C. castaneus, 3 to C. stigmaticus, and one to C. randalli. Of the 29 specimens sampled in Reunion, 27 were assigned to C. castaneus, one to C. stigmaticus, and one to C. randalli. Of the 5 Cirripectes samples from Rodrigues, one was assigned to C. castaneus, three to C. stigmaticus, and one to C. randalli. Photographs of the three species are presented in Appendix S12. Each species has a distinct color pattern: C. castaneus has orange to red vertical bars on the head and anterior body half, and orange-red colored upper and lower caudal fin rays, C. randalli has orange-red dots on the head and body, and C. stigmaticus has orange-red vertical bars on the head and dots on the anterior body half (Appendix S12). Their sizes varied from 29 to 67 mm with a median size of 37 mm for C. castaneus, 41-44 mm for C. randalli, and 34-47 mm for C. stigmaticus (Appendix S2).
Thus, C. stigmaticus (BOLD:AAE2834) was not genetically identified from Reunion prior to the present study (Table 4).

| Phylogeny of Cirripectes
Phylogenetic trees constructed on the COI gene were the most informative since the availability of the sequences of the alternative genes for the other species and localities was limited. The present study supplements the range of available markers for future analy-

ses. Phylogenetic trees corroborated the monophyly of the genus
Cirripectes. Despite some differences in internal branching order, the COI trees were generally congruent with the morphological phylogeny established by Williams (1988) and recent single marker phylogenies also based on the COI gene (Delrieu-Trottin et al., 2018;Hoban & Williams, 2020). The differences observed between our results and phylogenies produced by Hoban and Williams (2020) were among the short branches and poorly supported clades in both studies. The low resolution among these branches is probably due to multiple rapid divergence events with little time to accumulate shared mutations, even in a fast-evolving marker (Avise, 2009;Douzery, 2010).  variability, and a better taxon coverage for the genetic markers available, may resolve the remaining topological uncertainties or confirm the existence of fast diversification events.

| Molecular delimitation of Cirripectes
The number of clusters recovered by molecular species delineation approaches using the COI gene depends on the method used. In our case, the number of recovered clusters varied from 16 to 19. The ASAP delimitation approach generated less clusters than the treebased methods GMYC and mPTP, which is consistent with the literature (Dvořák et al., 2022;Kekkonen & Hebert, 2014;Puillandre et al., 2021). These results highlight the importance of performing multiple molecular approaches to determine the congruent clusters.
However, using multiple molecular markers (ideally from mitochondrial and nuclear DNA) combined with geographically wide sampling is needed to resolve remaining uncertainties. In addition, independent evidence, such as ecological data or morphological characters must be examined on all the specimens for integrative taxonomy and validation of the molecular species hypothesis (Puillandre et al., 2021). Our phylogeny and delimitation analyses also support the previous molecular distinction between Cirripectes alboapicalis, Cirripectes patuki sensu Delrieu-Trottin et al. (2018), and a new species (Delrieu-Trottin et al., 2018). Therefore, the use of the COI gene alone as a barcode could be sufficient for Cirripectes identification.

| Cirripectes in the Mascarene Islands
The type localities of two Cirripectes species are in the Mascarene Islands. The holotype of Salarias castaneus Valenciennes in Cuvier and Valenciennes (1836) was collected at Isle de France (Mauritius).
This species was later assigned to Cirripectes and, according to Williams (1988), several times erroneously synonymized with C. variolosus (e.g., Smith, 1959). The holotype of C. randalli Williams, 1988 originates from Cargados Carajos Shoals and paratypes come from Mauritius. Moreover, one paratype of C. gilberti Williams, 1988 is from Agalega. However, to date, few studies comprehensively assessed the diversity and distribution of Cirripectes species in the Mascarene archipelago.
Cirripectes occurrence at Agalega, Cargados Carajos Shoals, Mauritius, and Reunion was first described by Williams in Williams, 1988 (Table 3). C. castaneus and C. quagga were listed for Reunion, with the checklist subsequently completed based on visual records by Letourneur (1992) with C. polyzona by Fricke (1999) (1968) and Fricke (1999) as problematic, due to misidentifications and undocumented sight records (Heemstra et al., 2004). The Cirripectes sequences found in public databases were produced by studies not focused on Cirripectes but on taxonomically broad barcoding efforts of Indo-Pacific coral-reef fishes (Hubert et al., 2012) and post-larvae (Collet et al., 2017).
Both on Reunion and Rodrigues, C. castaneus, C. randalli, and C. stigmaticus were collected using ARMS. This means that ARMS sampled three out of the 6 Cirripectes species listed for Reunion and three out of four species listed for Rodrigues ( Table 4). These results were in agreement with the previous molecular studies available for Reunion, which reported C. castaneus and C. randalli (Collet et al., 2017;Hubert et al., 2015). To the best of our knowledge, we provide the first record of C. randalli for Rodrigues and the first sequence of C. stigmaticus for Reunion.
In spite of the extensive sampling using ARMS at Reunion (46 ARMS deployed at 10 sites in the course of 7 years), the absence of the other three species recorded for the island by visual censuses (C. filamentosus, C. polyzona, and C. quagga) can be explained by several hypotheses: (i) their habitats were not sampled, (ii) ARMS are not suitable to sample these species, (iii) these species are not present on the island, or (iv) were not present at the time of sampling.
The first possibility is that the sampling sites did not include the habitats of the species not sampled. Indeed, we sampled only on the top of spurs at 10-12 m depth, whereas some species (C. polyzona and C. quagga) are reported to prefer algal ridges and crests in the surf zone (Williams, 1988). Also, the public sequences of C. randalli were from specimens collected in tide pools or on shallow reef flats (JHB, pers. comm.). The difference in the habitat sampling may explain the low number of C. randalli recovered using ARMS on outer reef slopes.
The second explanation pertains to the sampling ability of ARMS for certain species. The ARMS are deployed on the reef surface, whereas other methods, such as rotenone or clove oil sampling, can potentially access cryptic species that live deeper within the reef matrix. In spite of earlier rotenone sampling at Reunion at various locations and depths (Hubert et al., 2012), only C. castaneus and C. randalli were recovered. In contrast, ARMS sampling allowed recovering an additional species, C. stigmaticus.
The third explanation rests on the hypothesis that the other three species do not occur on Reunion. From the first observation of Cirripectes, it has been reported that the species can be highly variable, leading to numerous misidentifications, even when morphology could be studied in detail (Smith, 1959;Williams, 1988).
Thus, visual surveys without detailed examination of the morphological characters may not permit to reliably identify all Cirripectes species. The species C. filamentosus, C. polyzona, and C. quagga listed for Reunion based exclusively on visual records might correspond to erroneous identifications. Ideally, the visual records of the "missing" species need to be confirmed by genetic analyses.
Fourth, Cirripectes, like other cryptobenthic reef fishes, are considered to have an abundant larval supply . It is therefore surprising that the "missing" three species have not been recovered during several years of sampling of post-larvae at Reunion

| Geographical ranges of Mascarene Cirripectes
The geographic ranges of the three Cirripectes species that we collected in the Mascarene Islands, C. castaneus, C. stigmaticus, and C. randalli, show different patterns. C. randalli was reported as an endemic species of the Mascarene Islands and listed in Mauritius, Cargados Carajos Shoals, and Reunion (Fricke, 1999;Williams, 1988).
The presence of C. randalli in Reunion confirmed in this study is consistent with the literature and previous molecular studies. To the best of our knowledge, this is the first report of C. randalli from Rodrigues, which is congruent with its known geographical range. Alternatively, given the recent highlight of cryptic species with small endemism areas in the genus (Delrieu-Trottin et al., 2018;Hoban & Williams, 2020), this could be the outcome of multiple cryptic lineages in the genus and in need of further investigation. Indeed, the presence of cryptic species could have implications for understanding mechanisms driving biodiversity patterns (Eme et al., 2018).
Cryptic species have distinct evolutionary patterns and, in some cases, a restricted geographical range with specialized behavior or higher threat susceptibility. Therefore, the detection of cryptic species is crucial for the conservation and/or management of marine biodiversity (Bickford et al., 2007).

| Extending and using DNA databases for specimen identification
It is of utmost importance to share molecular datasets that can be cross-checked and used openly for taxonomic or identification pur-  (Leis, 2015;Pentinsaari et al., 2020). In fact, even when authors are aware of this problem, the wrong assignation may occur.
Indeed, if a specimen is assigned to a BOLD BIN that contains specimens assigned to different species with no obvious errors about the geographic distribution, deciding which name is correct is problematic. Similar problems arise when a species corresponds to several BOLD BINs. To resolve these uncertainties, a new morphological examination of the specimens must be performed and, if needed, an investigation of phylogenetic relationships, possibly with added specimens and species (Ward et al., 2009).

| Primer selection and perspectives
Among the technical issues in molecular ecology, the choice of primer for PCR amplification is one of the most important factors affecting the probability of species detection. The eDNA approach led to the design of primers to respond to the new constraints, such as short ID sequences (<200 bp) to improve PCR success with degraded eDNA (Bylemans et al., 2018;Freeland, 2017). In the case of Cirripectes, two widely used primer pairs targeting the 12S were tested for comparison with results from the longer COI barcode.
Unlike the proprietary Teleo primers (Valentini et al., 2016), the MiFish primer set failed to amplify species in sillico, which contrasts with the results of Zhang et al. (2020) that showed that the latter primers had a larger detection range than the Teleo primer set. The genome areas targeted by COI and Teleo primers had a divergence >3% and thus allow automated species identification using the commonly used threshold for discriminating potential species. However, for the 12S targeted by the Teleo primers, only one sequence was available as a reference for this genus before our study, therefore, only the COI barcodes could truly be used for species identification.
In the future, primer limitations for eDNA surveys will be overcome with the development of shotgun sequencing for the eDNA, by direct sequencing of total eDNA and bypassing the PCR limitations associated with metabarcoding to provide insights into community composition (Taberlet et al., 2012;Tringe & Rubin, 2005). Currently, this approach is still limited by the completeness of the reference databases, in terms of species diversity (as barcoding) and in terms of reference genomes coverage (e.g., complete mitochondrial DNA).
Finally, multiplexed NGS sequencing of long amplicons produced cost-efficient complete mitogenomes, providing sequences for markers with greater variability than available previously. In the case of Cirripectes, ND1 and ND2 may be used instead of COI to resolve the remaining uncertainties. Moreover, using reads from NGS sequencing enables easy differentiation of alleles for nuclear markers.

| CON CLUS ION
In this study, the geographic distribution and species relationships within genus Cirripectes were examined. The major conclusions are writing -review and editing (equal).

ACK N OWLED G M ENTS
This study was supported by the research program 2AD) and help from its team. We are grateful to Gael Denys for his comments on the manuscript. Finally, we thank the reviewers for constructive comments on an earlier version of the manuscript.

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors state that they have no conflicting interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
All sequences produced in this study were deposited on GenBank